Extended-Spectrum β-Lactamase and carbapenemase-producing Escherichia coli O157:H7 among diarrheic patients in Shashemene, Ethiopia

Background The worldwide increase in multidrug resistance is a major threat to public health. One particular concern is the presence of Escherichia coli strains that carry Extended-Spectrum β-Lactamase (ESBL) and Carbapenemase enzymes, which can make multiple antibiotics ineffective. This complicates treatment strategies and raises the risk of illness and death. The aim of this study was to isolate E. coli O157:H7, assess its susceptibility against antimicrobial agents, and determine the presence of ESBL and Carbapenemase production in stool samples collected from diarrheic patients in Shashemene, west Arsi, Ethiopia from July to November 2022. Methods The samples were cultured McConkey Agar and E. coli were isolated and identified by standard biochemical tests using API 20E. E. coli O157:H7 was further identified using sorbitol McConkey Agar and antisera for O157 antigen test. The antimicrobial susceptibility test was performed using the Kirby–Bauer disc diffusion method using different antibiotics. Each identified isolate was screened and tested for phenotypical ESBL and Carbapenemase production using combined disc method and modified carbapenem inactivation method, respectively. Bivariant and multivariant analyses were employed using a logistic regression model for further analysis and were interpreted based on the odds ratio and level of statistical significance at a p-value <0.05 with 95% confidence interval. Results E. coli O157:H7 strain was found from 9% (38/423) study participants. The majority of the participants [61.9% (262/423)] were males; and 19.1% (81/ 423) of the participants were under five children. Living in urban areas, having domestic animals, and ≥5 family size in the household were identified as statistically significant factors associated with E. coli O157:H7. Twenty-seven (71.1%) and 12 (31.6%) of the 38 E. coli O157:H7 isolates were phenotypically confirmed to be ESBL and carbapenemase producers, respectively. All isolates were resistant against Ampicillin, but sensitive to ciprofloxacin. High resistance to Ampicillin and Amoxicillin/Clavulanic acid was observed among the ESBL and carbapenemase producing isolates also. The extent of detection of multidrug resistant E. coli O157:H7 isolates against three or more classes of antimicrobial agents tested was alarmingly very high (84%). Conclusion The E. coli O157:H7 isolates in this study showed a significant resistance to certain antimicrobials that were tested. The level of ESBL and Carbapenemase production among these isolates was found to be quite high. We observed a high resistance to Ampicillin and Amoxicillin/Clavulanic acid among the ESBL and carbapenemase producing isolates. Ciprofloxacin was found to be the most effective drug against both the ESBL producers and nonproducers.

Multidrug resistance has been increased all over the world, which is considered a public health threat.Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of antimicrobial susceptibility testing to detect the antibiotic of choice and the screening of the emerging Multidrug resistance (MDR) strains [10,11].
The emergence and global spread of antimicrobial resistance poses a significant threat to public health worldwide, especially in regions with limited resources and infrastructure for surveillance and control.Of particular concern are E. coli strains that carry Extended-Spectrum β-Lactamase (ESBL) and Carbapenemase enzymes, as they can render multiple antibiotics ineffective, complicating treatment strategies and increasing the risk of morbidity and mortality [12].
In the context of diarrheal diseases, the prevalence of ESBL and Carbapenemase-producing E. coli strains presents additional challenges in clinical management.These pathogens are resistant to commonly used antibiotics such as broad-spectrum β-lactams and carbapenems [13].This situation is particularly alarming in low-income countries like Ethiopia, where diarrheal diseases are endemic and healthcare facilities may have limited resources to combat antimicrobial resistance [14,15].ESBL and Carbapenemase production by Shiga toxin-producing E. coli was formerly uncommon.However, recently, there are some reports of ESBL and carbapenems-producing E. coli O157:H7 from different parts of the world.The first report of ESBL-producing E. coli O157:H7 was reported in Netherland from chicken isolates in 2003 [16] and the first report from a human clinical sample isolate was reported in Denmark, which originated from a fecal sample of a 2-year-old girl with bloody diarrhea who was part of a small outbreak in 2004.Until this year, there was no report of such scenario from human clinical isolates [17].There is also one study conducted in Japan that shows ESBL production among isolates found in beef cattle [18].Another study from Egypt found that 63.0% of E. coli O157:H7 isolated from meat and dairy products carry β-Lactamase-encoding genes [19].One study from Tanzania shows that 10% of isolate from cattle and 9.3% of isolate from human specimens have ESBL production capability [20].
To the best of our knowledge Even though there is little information regarding ESBL production in other parts of the world, there is no available published data regarding carbapenemase production of this bacteria in any part of the world.Regarding our country, it is similar to the world where no published data on ESBL and Carbapenamese production of this bacteria is available in Ethiopia.
The aim of this study was to investigate the prevalence, resistance to antimicrobials, and phenotypic characteristics of ESBL and Carbapenemase-producing E. coli O157:H7 strains among individuals suffering from diarrhea in Shashemene, Ethiopia.By acquiring a more comprehensive comprehension of the epidemiology and mechanisms of resistance within this specific context, valuable insights can be provided for local healthcare practices, initiatives on antimicrobial stewardship, and public health interventions that aim to control the dissemination of multidrug-resistant pathogens.Additionally, this research contributes to the global understanding of the dynamics of antimicrobial resistance and underscores the urgent requirement for coordinated strategies to address this escalating threat to human health.

Sample collection
This study was conducted in Shashemene Town and Shashemene Zuria Woreda under Ethiopian Cholera Control and Prevention (ECCP) project.The study site is selected because there were high number of diarrheal diseases reports from this area [21].This study was a prospective healthcare facility-based (HCF-based) cross-sectional type that included 8 public healthcare facilities (HCFs) (Abosto, Awasho, Chebi, Toga, Harbate, Fajogole health centers as well as Melka oda and Shashemene hospitals) found in Shashemene.A total of 41,563 patients visited all the selected healthcare facilities, of whom 1,399 patients were screened for eligibility requirements; and 1,388 of them fulfilled the inclusion criteria.Among these patients, 423 were selected systematically and enrolled in the study.Stool Sample collections were done from July 2022 to November 2022.All collected samples were tagged by sample ID, date of sampling, and sample type and then transported to the Shashemane Specialized Hospital Microbiology laboratory for microbiological analysis using a cold chain.

Bacterial isolation and identification
The stool sample were cultured and E. coli isolate were identified by colony characteristics on MacConkey agar (Condalab, Spain) as well as using Gram stain (Condalab, Spain), and API 20E (Biomerieux, France) standard biochemical tests.The isolates gave a distinct pink colony when growing on MacConkey agar due to ferementatio of lactose and colorless on Sorbitol MacConkey agar since this bacteria strain does not ferment sorbitol.Isolates also gave positive reaction for biochemical tests like Indole, catalase, lactose and glucose fermentation but negative for Urease production, Citrate utilization, hydrogen sulfide production, sorbitol fermentation and OxidaseFurther identification of E. coli O157:H7 was done using sorbitol utilization test on Sorbitol MacConkey agar (Condalab, Spain), and final serotyping was done using antisera for O157:H7 antigen (Denka, Japan).
Sample size calculation.The sample size required for this study is determined using a single proportions estimation method.In this method, the estimated proportion (P) is set at 50% because there hasn't been a previous large-scale investigation of E. coli O157:H7 among diarrheic patients from the general population As a result, the total sample size required for the study is calculated to be 427.

Extended Spectrum β-Lactamase production test
Phenotypic confirmatory tests for ESBL production were conducted by combined disk method using a third generation cephalosporins alone (cefotaxime, ceftriaxone, and ceftazidime) and in combination with clavulanic acid.A � 5 mm in zone diameter for either of antimicrobial agent tested in combination with clavulanate vs the zone diameter of the agent when tested alone was considered positive for ESBL production based on CLSI guidelines [22].

Carbapenemase production test
Phenotypic confirmatory tests for the ability of carbapenem-resistant isolates to produce Carbapenemase were investigated by the modified carbapenem inactivation method (MCIM) technique.A 1-μL loopful of the isolate to be tested was emulsified in 2ml trypticase soya broth (TSB) each and a 10-μg meropenem disk was immersed in each tube.After incubating for 4 hours ± 15 minutes the meropenem disk in the suspension was removed from each TSB-meropenem disk suspension and transferred to Mueller Hinton agar (MHA) plate already inoculated with E. coli ATCC 25922.The plate was then incubated at 35˚c ± 2˚c for 18 hours.Zone of inhibition of 6-15 mm and presence of pinpoint colonies within 16-18 mm zone were considered positive for carbapenemase production, according to CLSI guidelines [22].

Multidrug resistance bacteria
Based on CLSI guidelines we have considered E. coli O157:H7 isolates multidrug resistant (MDR) that acquired non-susceptibility to more than one class of antimicrobials.

Quality assurance
Prior to the data gathering procedure, stringent safeguards were put in place to assure data quality.WHO standardized data collection materials were used, which aided in the study's consistency and homogeneity.Furthermore, after each phase of preparation, the sterility of the media used for sample inoculation was extensively verified by incubating it overnight.The integrity of the samples and subsequent analyses were maintained by guaranteeing the sterility of the medium, reducing the likelihood of false-positive outcomes.A positive American Type Culture Collection (ATCC) was used to validate the functionality of each culture medium used.In addition, at each phase of confirmatory test for ESBL and carbapenemases, both positive and negative controls were included [Positive control (K.pneumoniae ATCC 700603 for ESBL) and (K.pneumoniae ATCC 1705 for Carbapenemase) and E. coli ATCC 25922 (negative control for both)].

Data management and statistical analysis
The data were coded and entered to SPSS version 27.0 software for analysis.Categorical covariates were summarized using frequencies and percentages, and numerical variables were summarized with a mean or median value based on the data distribution symmetry.Bi-variant analyses were employed using a logistic regression model and multi-variant analyses were employed using a logistic regression model for variables that have p-value �0.25in their bivariant analyses and further interpreted based on the odds ratio and level of statistical significance at a p-value <0.05 with 95% confidence interval.

Socio-demographic characteristics of the study participants
The median age of the study participants was 18 years with range of 1 to 75 years.Eighty-one (19.1%) of the participants were less than five years old and 14 (3.3%) of patients were elders aged above 55 years.The majority of the participants [262 (61.9%)] were males with a 1.6:1 male: female ratio.
Out of the 423 study participants, 258 (51.5%) lived in an urban area and more than half of the participants or guardians (in the case of children) [233 (55.1%)] attended only a primary school; and 126 (29.8%) study participants are farmers.In addition, the overwhelming majority of the participants [345 (81.6%)] had domestic animals.Tap water was the major source of drinking water for more than half of the participants [287 (67.8%)].The majority [291 (68.7%)] of the participants had a habit of eating uncooked food (Table 1).
Only those variables with P-value of �0.25 on their Bivariant analysis is incorporated in the table. https://doi.org/10.1371/journal.pone.0306691.t001

Discussion
In our study the presence of E. coli O157:H7 is found to be 9% of diarrheal cases.The isolated bacteria displayed resistance to various antimicrobial agents, but showed susceptibility to ciprofloxacin.In addition to that, phenotypic confirmation revealed that 71.1% of the bacterial isolates were ESBL producers, while 31.6% were carbapenemase producers and 26.3% isolates tend to produce both ESB and carbapenemase.All ESBL-producing and 92% carbapenemase producing isolates come from participants that own domestic animals but does not have significant association.
Our study also found that both adults and under-five children living in urban areas are susceptible to E. coli O157:H7 infection compared to those residing in rural areas, a finding which is in disagreement with another study conducted in eastern Ethiopia [26] which shows children living in a rural area were prone to E. coli O157:H7 infection than those living in an urban environments.The disparity could be attributed to factors such as higher population density, inadequate sanitation, reliance on food distribution networks, limited access to safe water, presence of animals, and urban lifestyle practices [29][30][31].
The prevalence of ESBL production is somewhat comparable to the findings from Egypt [63%, [19]] but much higher than the findings from Tanzania [9.3%, [20]].The difference in the distribution of ESBL producers could be because of variations in geographic location, study population, and differences in antibiotic prescribing practices and usage patterns between the research area in this study and the aforementioned locations.Different classes of antibiotics exert selective pressure, which can promote the establishment and spread of various ESBL genes.The use of antibiotics, including different types, dosages, and durations, can contribute to changes in the prevalence and distribution of ESBL genes in a specific area [13,32].As for the distribution of carbapenemase producer's since, this is the first study conducted to detect the carbapenemase production in E. coli O157:H7, we were unable to find any other studies to compare with our findings.The E. coli O157:H7 isolated in our study demonstrated significant antimicrobial resistance to Ampicillin [38/38 (100%)], Amoxicillin-clavulanic acid [34/38 (89.5%)],Tetracycline [22/38 (57.9%)], and an equal rate of 12/38 (31.6%) resistance to Ertapenem and Meropenem.However, all isolates exhibited sensitivity to ciprofloxacin.These findings align with those of previous studies conducted in Bahirdar [24], Ambo Town, [6], Jimma [33], Addis Ababa [34], and central Ethiopia [35], which reported high resistance rates to ampicillin and susceptibility rates above 93% to ciprofloxacin.Furthermore, 32 (84.2%) of the isolates demonstrated resistance to three or more classes of antimicrobials, consistent with the study conducted in Bahirdar which reported around 88% multidrug resistance.This high level of multidrug resistance raises concerns about therapeutic options, potentially resulting in treatment failures and increased morbidity/mortality rates.

Conclusions
During our laboratory investigation in Shashemene town and its surrounding areas, we found that 9% of diarrheal patients were infected with E. coli O157:H7 bacteria.Among these isolates, a high percentage (71.1%)produced ESBL, and 31.6%produced Carbapenemase.Fortunately, all isolates were susceptible to the antibiotic Ciprofloxacin, making it the preferred treatment option.However, in addition to ESBL and carbapenemase production, we observed an alarmingly high MDR rate of 84% among the E. coli O157:H7 strains in our study.These findings calls for a thorough epidemiological investigation on antimicrobial resistance that incorporates clinical, environmental and animal data across various sectors.By adopting such an approach, we can gain a deeper understanding of E. coli O157infections and antimicrobial resistance, especially concerning the sources of infection and their clinical consequences.

Fig 3 .
Fig 3. Magnitude of multi-drug resistant isolates.Keynote:-R1 = Resistance to one class of antimicrobial, R2 = Resistance to two class of antimicrobial, R3 = Resistance to three class of antimicrobial, R4 = Resistance to four class of antimicrobial, R5 = Resistance to five class of antimicrobial, R6 = Resistance to six class of antimicrobial, R7 = Resistance to seven class of antimicrobial, MDR = resistance to �3 class of antimicrobial.https://doi.org/10.1371/journal.pone.0306691.g003